Effects Of AGE-1on Regulation Of HCC Cell Viability, Migration, And Invasion Capacity And Gene Expressions In Vitro

Hepatocellular carcinoma (HCC) is the fifth most common diagnosed neoplasm in the world. Currently, China has the highest incidence rates of all reporting countries, accounting for about half of the annual new cases and deaths. Epidemiologic studies have indicated that HCC incidence rates are more than twice as high in males as in females, especially in China. To date, the etiology and molecular mechanisms responsible for HCC development remain to be defined, but epidemiological and experimental studies have identified several risk factors, including chronic hepatitis B or C virus (HBV or HCV) infection, aflatoxin B1exposure, heavy alcohol consumption, liver cirrhosis, and oral contraception. The risk associated with each of these factors varies widely among the world’s countries. For example, in China, where HBV is endemic, HBV infection is the predominant cause of HCC, while in the USA, where HBV is rare, alcohol-related cirrhosis is the major cause of HCC.Mechanistically, HCC development, like all other cancers, involves multiple genetic alterations, including oncogene activation and tumor-suppressor gene dysfunction. HBV DNA can insert into the human genome, causing genetic mutations or altered expressions that promote high rates of cell replication and decreased rates of apoptosis, both hallmarks of carcinogenesis. Over the past several decades, a large body of knowledge has been collected regarding the molecular alterations associated with HCC carcinogenesis, and several key signaling pathways, such as NF-κB, PI3K/AKT, MAPK, and Wnt/β-catenin, have been implicated in HCC development. Altered expression of the genes associated with these pathways has been shown to promote HCC cell proliferation, invasion, neoangiogenesis, and resistance to chemotherapy.Despite decades-long advances in screening for early stages of HCC (e.g. use of a-AFP as a tumor marker) and improvement in treatments (e.g. liver transplantation), survival of HCC patients is still very low because HCC continues to be diagnosed at later clinical stages and the treatment options are not curative. The existence of metastatic HCC or poor general health precludes surgical resection of HCC lesions However, even in cases of successful tumor resection, a certain percentage of HCC may metastasize to other organs and the recurrence rate is up to70%within5-years after surgery. Therefore, a better understanding of HCC biology is urgently needed to aid in development of innovative approaches for HCC treatment and prevention.Astrocyte elevated gene-1(AEG-1) is an oncogene, recently identified from HIV-infected or TNF-α-treated primary human fetal astrocytes. To date, various studies using immunohistochemistry have demonstrated that AEG-1is overexpressed in a number of tumor tissues, including HCC, breast cancer, prostate cancer, gastric cancer, real cancer, colorectal cancer, esophageal cancer, small cell lung cancer, melanoma, and glioma. AEG-1was reported to be positive in93.6%of HCC tissue specimens. Experimental analyses of AEG-1gene knock-in models or AEG-1silencing using small interfering (si)RNA molecules showed that AEG-1protein plays an important role in tumor formation, proliferation, invasion, metastasis, angiogenesis, and resistance to chemotherapyFunctionally, AEG-1is the downstream gene of Ha-ras. Ha-ras is known to activate PI3K/Akt/GSK3p, thereby leading to c-Myc binding to AEG-1protein and subsequent up-regulation of AEG-1expression or activation of NF-κB, MAPK, and Wnt/β-catenin signaling pathways. Furthermore, LY294002is a specific PI3K inhibitor that suppresses PI3K/AKT-induced AEG-1expressionThis study investigated the effects of PI3K inhibitor, LY294002, on suppression of astrocyte elevated gene-1(AEG-1) for regulation of HCC cell viability, apoptosis, and invasion in vitro. Cell lines derived from normal liver and HCC were treated with LY294002and evaluated by RT-PCR, Western blot, MTT, Immunocytochemistry, Millicell assays. The data showed that AEG-1mRNA and protein were overexpressed in HCC cells, compared to the normal liver cells. LY294002treatment of HCC cells significantly reduced tumor cell viability, but promoted apoptosis. Tumor cell migration and invasion assays showed that LY294002treatment also decreased the capacity of HCC cell migration and invasion. Molecularly, LY294002treatment reduced AEG-1expression, AKT and GSK3β phosphorylation, and expression of cyclinD1, CDK4, VEGF as well as Bcl2, but up-regulated Bax and c-Myc expression.The data from this study demonstrated usefulness of AEG-1inhibitor, LY294002, for effective control of HCC.Despite all of these promising findings, this in vitro study is considered preliminarily and more work needs to be done before small size AEG-1inhibitor can be translated into clinical practice to treat HCC patients. In future studies, we plan to test effects of AEG-1inhibitor, LY294002, in an in vivo model system, such as nude mouse xenograft assay. We will also investigate the defined molecular mechanism by which AEG-1regulates other gene pathways beyond PI3K-AEG-1signaling. Regardless, this study demonstrated a clear target of LY294002in HCC cells, suggesting its potential usefulness in clinical treatment of HCC.